In Vitro Cell-Based MTT and Crystal Violet Assays for Drug Toxicity Screening.

The development of novel drug candidates is a current challenge in pharmacology where therapeutic benefits must exceed side effects. Toxicology testing is therefore a fundamental step in drug discovery research. Herein, we describe the first line of toxicology testing program, consisting in cell-based high-throughput screening assays, which have the advantage of being easy, rapid, cheap, and reproducible while providing quantitative information. We illustrate MTT and Crystal Violet assays, two common colorimetric tests able to assess both cytostatic and cytotoxic effects, respectively, of a drug candidate. MTT assay allows evaluation of cellular metabolic activity, by which cell viability can be inferred; Crystal Violet staining is directly correlated with attached viable cells, thus allowing direct assessment of cell survival and death. Therefore, combination of the two methodologies represents a useful tool for predicting drug sensitivity and efficacy, the first milestones in pre-clinical toxicology workflow.

γδ T-Cell Immunophenotype for the Study of Human Aging.

Flow cytometry serves as a crucial tool in immunology, allowing for the detailed analysis of immune cell populations. γδ T cells, a subset of T cells, play pivotal roles in immune surveillance and immune aging. Assessing the phenotype and functional capabilities of γδ T cells isolated from whole blood or tissue within the context of human aging yields invaluable insights into the dynamic changes affecting immune function, tissue homeostasis, susceptibility to infections, and inflammatory responses.

Decoding biology with massively parallel reporter assays and machine learning.

Massively parallel reporter assays (MPRAs) are powerful tools for quantifying the impacts of sequence variation on gene expression. Reading out molecular phenotypes with sequencing enables interrogating the impact of sequence variation beyond genome scale. Machine learning models integrate and codify information learned from MPRAs and enable generalization by predicting sequences outside the training data set. Models can provide a quantitative understanding of cis-regulatory codes controlling gene expression, enable variant stratification, and guide the design of synthetic regulatory elements for applications from synthetic biology to mRNA and gene therapy. This review focuses on cis-regulatory MPRAs, particularly those that interrogate cotranscriptional and post-transcriptional processes: alternative splicing, cleavage and polyadenylation, translation, and mRNA decay.

Protocol to calculate the synergy of drug combinations in organoids and primary cells from murine tumors.

Evaluating the synergy of drug combinations is crucial in advancing treatment regimens. Here, we present a protocol to establish primary cells and organoids from murine tumors and calculate drug synergy. We describe all necessary cell culture procedures, including establishing primary cultures, setting up treatment groups, and detecting cell viability. We then outline how to calculate the synergy score based on a bioinformatical pipeline. This approach applies to any disease model in which a combination of drugs needs to be evaluated. For complete details on the use and execution of this protocol, please refer to Ku et al.1.

Protocol for detecting endogenous GPCR activity in primary cell cultures using ONE-GO biosensors.

ONE vector G protein Optical (ONE-GO) biosensors can measure the activity of endogenously expressed G protein-coupled receptors (GPCRs) in primary cells. By detecting G proteins that belong to all four families (Gs, Gi/o, Gq/11, G12/13) across cell types, these biosensors provide high experimental versatility. We first describe steps to express ONE-GO biosensors in primary cells using lentiviral transduction. We then detail how to carry out measurements and subsequent analysis to quantify changes in bioluminescence resonance energy transfer (BRET) reporting on endogenous GPCR activity. For complete details on the use and execution of this protocol, please refer to Janicot et al.1.

Biocatalytic Ether Lipid Synthesis by an Archaeal Glycerolprenylase.

Although ethers are common in secondary natural products, they are an underrepresented functional group in primary metabolism. As such, there are comparably few enzymes capable of constructing ether bonds in a general fashion. However, such enzymes are highly sought after for synthetic applications as they typically operate with higher regioselectivity and under milder conditions than traditional organochemical approaches. To expand the repertoire of well characterized ether synthases, we herein report on a promiscuous archaeal prenyltransferase from the scarcely researched family of geranylgeranylglyceryl phosphate synthases (GGGPSs or G3PSs). We show that the ultrastable Archaeoglobus fulgidus G3PS makes various (E)- and (Z)-configured prenyl glycerol ethers from the corresponding pyrophosphates while exerting perfect control over the configuration at the glycerol unit. Based on experimental and computational data, we propose a mechanism for this enzyme which involves an intermediary prenyl carbocation equivalent. As such, this study provides the fundamental understanding and methods to introduce G3PSs into the biocatalytic alkylation toolbox.

Effects of temperature and storage duration on quality of an insect artificial diet.

Artificial diets are widely used to produce insects for research and education programs. Completed diets, in which the diets are fully made from individual ingredients and ready to use, often have high water activity, making them vulnerable to degradation. Proper storage is critical to maintaining diet quality, yet the storage conditions are not well investigated. In this study, we characterized the effects of storage conditions (temperatures and storage duration) on the quality of a diet capable of rearing both specialist and generalist insect species. The completed diet, produced by both private industry and a USDA-Agricultural Research Service laboratory, was exposed to varying temperatures during a 24-hour transit over 1600 km. After transit, it was stored at 4°C for a total storage period of 28 days. In a separate experiment, the completed diet was stored immediately after diet production at five fixed temperatures (-20, 4, 22, 25, and 33°C) for up to 28 days. For both experiments, at 5 intervals after storage (1, 7, 14, 21, and 28 days), diet quality was accessed by life history parameters (survival, molting, and weight) of western corn rootworm (Diabrotica virgifera virgifera LeConte) larvae, the most serious maize pest in the United States. Our results showed that exposure to varying temperatures between -2°C and 27°C for 24 hours had no significant impact on diet quality. However, extended storage (beyond 24 hours) at any of the fixed temperatures negatively affected diet quality. Insects reared on diets stored for over 24 hours at fixed temperatures ranging from -20°C to 33°C had significant declines in performance. Among the tested temperatures, -20°C and 4°C were found to be the most effective for preserving diet quality. At these low temperatures, there were no significant changes in insect weight and survival for diets stored within 21 and 28 days, respectively, though molting was significantly reduced within 7 days of storage. These findings provide the base of information on the storage conditions for completed diets, supporting the production of healthy insects.

Antiviral drugs in animal-derived matrices: A review.

The ban of antiviral drugs in food-producing animals in several parts of the world, latest by Commission Delegated Regulation (EU) 2022/1644, has increased the need for food control laboratories to develop analytical methods and perform official controls. However, little is known about antiviral drugs, their use, and its analysis in food-producing animals in the EU. This review aims to provide insights into relevant viruses, antiviral drugs, and animal-derived matrices for analytical method development and monitoring purposes to implement in food control laboratories. For years, animal viruses, such as African swine fever and avian influenza, have caused many outbreaks. Besides, they led to large economic losses due to the applied control measures and a lack of available treatments. Considering these losses and the known effectiveness of authorized human antiviral drugs in different organisms, medicines such as amantadine in Chinese poultry have been misused. Various analytical methods, including screening assays and sensors (published 2016-2023), and mass spectrometry methods (published 2012-2023) have been outlined in this review for the monitoring of antiviral drugs in animal-derived matrices. However, pharmacokinetics information on metabolite formation and distribution of these substances in different animal-derived matrices is incomplete. Additionally, apart from a few countries, there is a lack of available data on the potential misuse of different antiviral drugs in animal-derived matrices. Although a handful of important antiviral drugs, such as influenza, broad-spectrum, antiretroviral, and herpes drugs, and animal-derived matrices, such as chicken muscle, are identified, the priority of the scope should be further specified by closing the aforementioned gaps.

Tuberculosis in kidney transplant candidates and recipients.

Tuberculosis (TB) is the leading cause of infectious mortality and morbidity in the world, second only to coronavirus disease 2019. Patients with chronic kidney disease and kidney transplant recipients are at a higher risk of developing TB than the general population. Active TB is difficult to diagnose in this population due to close mimics. All transplant candidates should be screened for latent TB infection and given TB prophylaxis. Patients who develop active TB pre- or post-transplantation should receive multidrug combination therapy of antitubercular therapy for the recommended duration with optimal dose modification as per glomerular filtration rate.

In vivo antischistosomal efficacy of Porcelia ponderosa γ-lactones.

BACKGROUND: Schistosomiasis, caused by the parasitic blood fluke Schistosoma mansoni, is a significant global health concern, particularly in tropical and subtropical regions. The available chemotherapeutic drug is restricted to praziquantel with present problems related to efficacy, toxicity and resistance, justifying the search for new drugs. Different natural products, including γ-lactones, have demonstrated anthelmintic activity. Thus, in this study, new γ-lactones from Porcelia ponderosa were investigated for their anti-S. mansoni effects in vitro and in vivo. PURPOSE: To evaluate the therapeutical potential against S. mansoni of the mixture of γ-lactones 1 + 2 obtained from Porcelia ponderosa seeds. STUDY DESIGN AND METHODS: The precipitate formed during the concentration of CH2Cl2 extract from seeds of P. ponderosa showed to be composed by a mixture of the new γ-lactones 1 + 2 (in a ratio 77:23) which were chemically characterized using NMR and ESI-HRMS. This mixture was evaluated in vitro and in vivo against S. mansoni, using a murine model of schistosomiasis. Additionally, toxicity of the mixture of 1 + 2 (77:23) was determined using mammalian cell lines (in vitro) or the model organism Caenorhabditis elegans (in vivo). RESULTS: Seeds of P. ponderosa afforded a mixture of two unreported γ-lactones, 3­hydroxy-4-methylene-2-(tetracosa-17'Z,23'-diene-13',15'-diynyl)­but-2-enolide (1) and 3­hydroxy-4-methylene-2-(tetracos-17'Z-ene-13',15'-diynyl)­but-2-enolide (2). Initially, the antischistosomal activity of the mixture of 1 + 2 (77:23) was investigated in vitro, and obtained results demonstrate reduced activity against Schistosoma mansoni worms (EC50 of 83.3 µg/ml) in comparison to positive control praziquantel (EC50 of 1.5 µg/ml). However, when tested in vivo using oral administration at 400 mg kg-1, the standard dose used in the murine model of schistosomiasis, the mixture of 1 + 2 (77:23) revealed expressive reductions in both worm burden (65.7 %) and egg production (97.2 %), similar of those observed to praziquantel (89.7 % and 91.5 %, respectively). On the other hand, when treated using 200 and 100 mg kg-1, reductions in worm burden (25.7 and 12.4 %) and egg production (33.6 and 13.3 %) were also observed. Importantly, the mixture of 1 + 2 (77:23) exhibited no toxicity using mammalian cell lines (in vitro) or C. elegans (in vivo). CONCLUSION: Considering the promising in vivo activity of γ-lactones from P. ponderosa, the mixture of 1 + 2 (77:23) can be considered as promising candidate for the development of novel antischistosomal therapeutics, underscoring the importance of biodiversity exploration in the search for effective treatments against neglected tropical diseases.

Saturation mutagenesis-reinforced functional assays for disease-related genes.

Interpretation of disease-causing genetic variants remains a challenge in human genetics. Current costs and complexity of deep mutational scanning methods are obstacles for achieving genome-wide resolution of variants in disease-related genes. Our framework, saturation mutagenesis-reinforced functional assays (SMuRF), offers simple and cost-effective saturation mutagenesis paired with streamlined functional assays to enhance the interpretation of unresolved variants. Applying SMuRF to neuromuscular disease genes FKRP and LARGE1, we generated functional scores for all possible coding single-nucleotide variants, which aid in resolving clinically reported variants of uncertain significance. SMuRF also demonstrates utility in predicting disease severity, resolving critical structural regions, and providing training datasets for the development of computational predictors. Overall, our approach enables variant-to-function insights for disease genes in a cost-effective manner that can be broadly implemented by standard research laboratories.

Experimental Considerations for the Evaluation of Viral Biomolecular Condensates.

Biomolecular condensates are nonmembrane-bound assemblies of biological polymers such as protein and nucleic acids. An increasingly accepted paradigm across the viral tree of life is (a) that viruses form biomolecular condensates and (b) that the formation is required for the virus. Condensates can promote viral replication by promoting packaging, genome compaction, membrane bending, and co-opting of host translation. This review is primarily concerned with exploring methodologies for assessing virally encoded biomolecular condensates. The goal of this review is to provide an experimental framework for virologists to consider when designing experiments to (a) identify viral condensates and their components, (b) reconstitute condensation cell free from minimal components, (c) ask questions about what conditions lead to condensation, (d) map these questions back to the viral life cycle, and (e) design and test inhibitors/modulators of condensation as potential therapeutics. This experimental framework attempts to integrate virology, cell biology, and biochemistry approaches.

Reaction Times in Modern Virology: "This is not a Drill".

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant of interest BA.2.87.1 has not driven any Coronavirus disease 2019 (COVID-19) pandemic wave. Nevertheless, it has served to test the reaction times of modern virology laboratories. In this commentary, we highlight how fast the reaction has been at characterizing this sublineage, leading at an unprecedented pace to almost as many papers as the number of viral sequences.

Levels of Amyloid Beta (Aß) Expression in the Caenorhabditis elegans Neurons Influence the Onset and Severity of Neuronally Mediated Phenotypes.

A characteristic feature of Alzheimer's disease (AD) is the formation of neuronal extracellular senile plaques composed of aggregates of fibrillar amyloid ß (Aß) peptides, with the Aß1-42 peptide being the most abundant species. These Aß peptides have been proposed to contribute to the pathophysiology of the disease; however, there are few tools available to test this hypothesis directly. In particular, there are no data that establish a dose-response relationship between Aß peptide expression level and disease. We have generated a panel of transgenic Caenorhabditis elegans strains expressing the human Aß1-42 peptide under the control of promoter regions of two pan-neuronal expressed genes, snb-1 and rgef-1. Phenotypic data show strong age-related defects in motility, subtle changes in chemotaxis, reduced median and maximum lifespan, changes in health span indicators, and impaired learning. The Aß1-42 expression level of these strains differed as a function of promoter identity and transgene copy number, and the timing and severity of phenotypes mediated by Aß1-42 were strongly positively correlated with expression level. The pan-neuronal expression of varying levels of human Aß1-42 in a nematode model provides a new tool to investigate the in vivo toxicity of neuronal Aß expression and the molecular and cellular mechanisms underlying AD progression in the absence of endogenous Aß peptides. More importantly, it allows direct quantitative testing of the dose-response relationship between neuronal Aß peptide expression and disease for the first time. These strains may also be used to develop screens for novel therapeutics to treat Alzheimer's disease.

Diagnosis and Management of Neonatal Bacterial Sepsis: Current Challenges and Future Perspectives.

Sepsis remains the second cause of death among neonates after the pathological consequences of extreme prematurity. In this review we summarized knowledge about pathogens causing early-onset sepsis (EOS) and late-onset sepsis (LOS), the role of perinatal risk factors in determining the EOS risk, and the tools used to reduce unnecessary antibiotics. New molecular assays could improve the accuracy of standard blood cultures, providing the opportunity for a quick and sensitive tool. Different sepsis criteria and biomarkers are available to date, but further research is needed to guide the use of antibiotics according to these tools. Beyond the historical antibiotic regimens in EOS and LOS episodes, antibiotics should be based on the local flora and promptly modulated if specific pathogens are identified. The possibility of an antibiotic lock therapy for central venous catheters should be further investigated. In the near future, artificial intelligence could help us to personalize treatments and reduce the increasing trend of multidrug-resistant bacteria.

Beyond basic characterization and omics: Immunomodulatory roles of platelet-derived extracellular vesicles unveiled by functional testing.

Renowned for their role in haemostasis and thrombosis, platelets are also increasingly recognized for their contribution in innate immunity, immunothrombosis and inflammatory diseases. Platelets express a wide range of receptors, which allows them to reach a variety of activation endpoints and grants them immunomodulatory functions. Activated platelets release extracellular vesicles (PEVs), whose formation and molecular cargo has been shown to depend on receptor-mediated activation and environmental cues. This study compared the immunomodulatory profiles of PEVs generated via activation of platelets by different receptors, glycoprotein VI, C-type lectin-like receptor 2 and combining all thrombin-collagen receptors. Functional assays in vivo in zebrafish and in vitro in human macrophages highlighted distinct homing and secretory responses triggered by the PEVs. In contrast, omics analyses of protein and miRNA cargo combined with physicochemical particle characterization found only subtle differences between the activated PEV types, which were insufficient to predict their different immunomodulatory functions. In contrast, constitutively released PEVs, formed in the absence of an exogenous activator, displayed a distinct immunomodulatory profile from the receptor-induced PEVs. Our findings underscore that PEVs are tunable through receptor-mediated activation. To truly comprehend their role(s) in mediating platelet functions among immune cells, conducting functional assays is imperative.

Click-chemistry-based protocol for detecting 4-octyl-itaconate-alkylated proteins in primary mouse macrophages.

4-Octyl itaconate (4-OI), a derivative of itaconate, inhibits inflammation by alkylating its target proteins. Here, we present a click-chemistry-based protocol for detecting 4-OI-alkylated proteins in mouse primary bone-marrow-derived macrophages (BMDMs) by using an itaconate-alkyne (ITalk) probe. We describe steps for culturing and treating BMDMs and details on using click chemistry in the cell lysate. We also detail procedures for detecting alkylated proteins by western blot. For complete details on the use and execution of this protocol, please refer to Su et al.1.

Protocol for lipid mediator profiling and phenotyping of human M1- and M2-monocyte-derived macrophages during host-pathogen interactions.

Here, we present a protocol for primary, human immune cell isolation and stimulation for lipid mediator profiling. We describe steps for the isolation of monocytes from human leukocyte concentrates via density centrifugation and differentiation/polarization toward M1- or M2-monocyte-derived macrophages (MDMs). We detail stimulation approaches of MDMs with live bacteria or influenza A virus for lipid mediator profiling and sample preparation for subsequent analysis, such as enzyme expression, mRNA analysis, or surface marker determination. For complete details on the use and execution of this protocol, please refer to Jordan et al.1.

Genome-Wide Survey of the Potential Function of CrLBDs in Catharanthus roseus MIA Biosynthesis.

Catharanthus roseus (C. roseus) can produce over 150 types of monoterpenoid indole alkaloids (MIAs), including vinblastine and vincristine, which are currently the primary sources of these alkaloids. Exploring the complex regulatory mechanisms of C. roseus is significant for resolving MIA biosynthesis. The Lateral Organ Boundaries Domain (LBD) is a plant-specific transcription factor family that plays crucial roles in the physiological processes of plant growth, stress tolerance, and specialized metabolism. However, the LBD gene family has not been extensively characterized in C. roseus, and whether its members are involved in MIA biosynthesis is still being determined. A total of 34 C. roseus LBD (CrLBD) genes were identified. RNA-Seq data were investigated to examine the expression patterns of CrLBD genes in various tissues and methyl jasmonate (MeJA) treatments. The results revealed that the Class Ia member CrLBD4 is positively correlated with iridoid biosynthetic genes (p < 0.05, r ≥ 0.8); the Class IIb member CrLBD11 is negatively correlated with iridoid biosynthetic genes (p < 0.05, r ≤ -0.8). Further validation in leaves at different growth stages of C. roseus showed that CrLBD4 and CrLBD11 exhibited different potential expression trends with iridoid biosynthetic genes and the accumulation of vindoline and catharanthine. Yeast one-hybrid (Y1H) and subcellular localization assays demonstrated that CrLBD4 and CrLBD11 could bind to the "aattatTCCGGccgc" cis-element and localize to the nucleus. These findings suggest that CrLBD4 and CrLBD11 may be potential candidates for regulating MIA biosynthesis in C. roseus. In this study, we systematically analyzed the CrLBD gene family and provided insights into the roles of certain CrLBDs in the MIA biosynthesis of C. roseus.

Development of Novel Alaninamide Derivatives with Anticonvulsant Activity and Favorable Safety Profiles in Animal Models.

In our current study, we developed a focused series of original ((benzyloxy)benzyl)propanamide derivatives that demonstrated potent activity across in vivo mouse seizure models, specifically, maximal electroshock (MES) and 6 Hz (32 mA) seizures. Among these derivatives, compound 5 emerged as a lead molecule, exhibiting robust protection following intraperitoneal (i.p.) injection, as follows: ED50 = 48.0 mg/kg in the MES test, ED50 = 45.2 mg/kg in the 6 Hz (32 mA) test, and ED50 = 201.3 mg/kg in the 6 Hz (44 mA) model. Additionally, compound 5 displayed low potential for inducing motor impairment in the rotarod test (TD50 > 300 mg/kg), indicating a potentially favorable therapeutic window. In vitro toxicity assays further supported its promising safety profile. We also attempted to identify a plausible mechanism of action of compound 5 by applying both binding and functional in vitro studies. Overall, the data obtained for this lead molecule justifies the more comprehensive preclinical development of compound 5 as a candidate for a potentially broad-spectrum and safe anticonvulsant.